Bi-specific conjugates

a conjugate and bi-specific technology, applied in the field of new drugs, can solve the problems of poor efficacy of cd40 agonists in leading to effective t-cell activation, insufficient cd40 stimulation for t-cell activation, and inability to always be antigenic materials, so as to facilitate the preparation of patient-specific therapeutic agents and improve the efficacy of complexes. effect and cost

Pending Publication Date: 2022-01-06
STRIKE PHARM AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a new type of molecule that can be used in personalized medicine. It consists of two different parts that are connected together. One part is specific for a protein called CD40, which is found on antigen-presenting cells. The other part is specific for a tag, which is attached to an antigen. When the two parts bind to each other, they form a complex that can activate the CD40 protein and also present the antigen to the immune system. This design allows for flexibility in creating these molecules for individual patients, as only different tags need to be prepared, which is easier and cheaper than creating separate molecules for each patient. The molecule is also more effective at stimulating the immune system compared to other methods.

Problems solved by technology

However, antigenic material may not always be present (for example if a tumour has been resected), and CD40 agonists may have poor efficacy in leading to effective T-cell stimulation in such a situation.
CD40 stimulation may also be insufficient for T-cell activation (for example if there is a dose-limiting toxicity of the CD40 agonist as an infusion product).

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Materials and Methods

Cells

[0255]B3Z (Karttunen et al., PNAS 89(13): 6020-6024, 1992), a murine T-cell hybridoma expressing a TCR which recognises the ovalbumin peptide OVA257-264 (SIINFEKL, SEQ ID NO: 1) in the context of the murine Class I MHC H-2Kb, was used to assess peptide loading and subsequent antigen presentation. B3Z cells express β-galactosidase under the control of the IL-2 promoter and thus, upon T-cell activation and proliferation, the enzyme will be expressed. β-galactosidase is able to hydrolyse the substrate chlorophenol red-β-D-galactopyranoside (CPRG), which leads to a colour change with a magnitude corresponding to the level of B3Z T-cell activation. Hence B3Z activation may be detected and measured by spectrophotometry.

[0256]Pmel-1 mice (Jackson Laboratory (USA), mouse strain 005023, described further below) are transgenic mice with T-cells which express a TCR specific for the murine gp100(25-33) peptide (SEQ ID NO: 2, amino acids 25-33 of the melanoma antigen gp...

example 2

Spread of T-Cell Activation Following Therapeutic Administration

Materials and Methods

[0289]Adult female C57BL / 6 mice (18-20 g weight) were administered CFSE (carboxyfluorescein succinimidyl ester)-labelled splenocytes from Pmel-1 mice and tghCD40 immature BMDC as described in Example 1 above (see “In Vivo Experimental Setup”). On day 1, various combinations of antibodies and UU-30 peptide were administered as described above. The antibodies used in this example were Ab-2 and Bi-10, which were administered at doses of 7.5 pmol (low, L); 15 pmol (medium, M); and 22.5 pmol (high, H). The UU-30 peptide was administered at doses of 18.75 pmol (L), 37.5 pmol (M) and 56.25 pmol (H).

[0290]After 72 hours, the draining popliteal and non-draining inguinal lymph nodes were harvested and passed through a 70 μm cell strainer to obtain single cell suspensions. Pmel-1 cell accumulation and proliferation was assessed by flow cytometry. Pmel-1 T-cells were gated out based on expression of the congeni...

example 3

of the Interaction Between a Tag and an scFv on a Tetravalent Antibody

Materials and Methods

[0292]The bispecific antibody Bi-17 contains the FITC-8 scFv, which recognises FITC. The scFv is located at the C-terminus of the heavy chain of the anti-CD40 antibody (i.e. it is encoded C-terminal to the IgG2 constant region). The amino acid sequence of the FITC-8 scFv is set forth in SEQ ID NO: 70.

[0293]The Bi-17 bispecific antibody was produced by Absolute Antibody (UK). The antibody was expressed in HEK293 cells, and purified by affinity chromatography using protein A followed by preparative size exclusion chromatography (SEC). The purity was determined by SDS-PAGE to be >98% and monomeric content determined to be 94% by analytical SEC. Endotoxin levels were <1 EU / mg as determined by LAL chromogenic endotoxin assay.

CD14 Monocyte and PBMC Isolation.

[0294]Peripheral blood mononuclear cells (PBMCs) were isolated from Buffy Coats, donated by healthy volunteers, by Ficoll separation using SepM...

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Abstract

The present invention provides a conjugate comprising: i) at least one first specific binding molecule which binds CD40, wherein said first specific binding molecule is an agonist of CD40; and ii) at least one second specific binding molecule which binds a tag moiety, wherein said tag moiety is not a cancer antigen, wherein said first specific binding molecule and second specific binding molecule are antigen-binding proteins comprising an antigen-binding domain of an antibody and are covalently linked. The conjugate can be combined with a tag construct comprising: i) a tag moiety which is not a cancer antigen; and ii) an antigen, being a cancer antigen or an antigen derived from a pathogen; wherein said antigen is a polypeptide and said tag moiety is covalently linked to said antigen, for use in therapy, to stimulate an immune response by a subject against the antigen in question.

Description

FIELD OF THE INVENTION[0001]The present invention relates to novel conjugates comprising a first binding molecule capable of binding specifically to CD40 conjugated, via a covalent linkage, including particularly a peptide bond, to a second binding molecule capable of binding to a tag moiety to form a conjugate-tag complex. The tag moiety may be provided as part of a tag construct further comprising an antigen. The invention further relates to a complex of the conjugate with such a tag-antigen construct, and the use of such a complex in therapy, particularly for the treatment of cancer.BACKGROUND TO THE INVENTION[0002]Monoclonal antibodies (mAbs) which modulate immune responses are proving highly effective in cancer treatment, with increasing evidence that such responses can be harnessed to provide durable eradication of tumours. Various antibodies against different targets have been developed, e.g. targeting the immune checkpoints CTLA-4 and PD-1, which support the view that T-cell...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/08A61K39/385A61K39/39A61K39/395C07K16/12C07K16/28
CPCA61K39/08A61K39/385A61K39/39A61K39/39541A61K2039/627C07K16/2878A61K39/39558A61K39/3955C07K16/1282A61K2039/6056A61K2039/622A61K2039/64C12N2710/16134A61K39/12C07K14/33C07K2319/40C07K2317/31C07K2317/77C07K2317/64C07K2319/31A61K39/4615A61K2239/57A61K39/4622A61K2239/31A61K2239/38A61K39/461A61K39/4611A61K39/464492A61P35/00A61P31/12A61K2039/507
Inventor MANGSBO, SARAPERSSON LOTSHOLM, HELENA
Owner STRIKE PHARM AB
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